Abstract The nucleus accumbens shell (NAcSh) receives glutamatergic projections from several limbic and paralimbic brain regions, with each projection presumably conveying different aspects of emotional and motivational arousals. In motivated behaviors, these excitatory inputs are typically activated concurrently, sending converging excitatory inputs to medium spiny neurons (MSNs), principal neurons in the NAcSh. While recent techniques allow for dissecting individual NAcSh projections, it remains largely unknown whether different NAcSh projections interact with each other when co-activated, and, if so, what the anatomical basis underlies these interactions. To start to address these knowledge gaps, this R21 application focuses on two prominent glutamatergic inputs to the NAcSh, the projections from the basolateral amygdala (BLAp) and paraventricular nucleus of the thalamus (PVTp). Both BLAp and PVTp form monosynaptic contacts onto NAcSh MSNs, but they are differentially involved in NAcSh-based behaviors. With the dual-rhodopsin expression system controlled by tow lasers with different wavelengths, we can simultaneously and independently activate BLAp and PVTp synaptic transmission to the same MSNs. The preliminary results show that a brief co-activation of these two projections induced a short-term potentiation in BLAp transmission but a short-term depression in PVTp transmission. Thus, co-activation temporarily boosted the informational flow through the BLAp while constrained the informational flow through the PCTp. These results not only indicate a clear functional interaction between the BLAp and PVTp, but also provide a potential circuit mechanism through which some motivational arousals override others under certain conditions. The first objective of this application is to extend these preliminary findings by exploring how BLAp and PVTp synapses functionally interact with each other on NAcSh MSNs. Using the dual-color SynapTag technique combined with postsynaptic filling, the second objective of this application is to determine the anatomical arrangement of the BLAp and PVTp that confers the functional interaction of these two projections. Outcomes of the proposed experiments may provide essential functional and anatomical mechanisms through which different aspects of emotional and motivational arousals interact and coordinate for behavioral prioritization.